The use of optical measuring devices, for instance in research and development or in industrial quality control, presents a great many problems, which require regular calibration and characterization of optical measuring devices. These are to be illustrated by way of the example of luminescence-measuring instruments, where reference systems for the measurement of intensities are additionally required.
Spectral Fluorescence and Emission Standards for Emission Correction
Every luminescence-measuring technique supplies measurement data that are composed of analyte- and instrument-specific contributions. These reflect the wavelength dependence of the light source(s) and optical structural elements contained in the excitation and emission channel of the instrument, as well as the spectral sensitivity of the detection systems used. The comparability of luminescence data over instrument and laboratory limits, the determination of instrument aging, the restorability (EN ISO/IEC 17025) of luminescence data and the optimization of luminescence methods require the determination of these instrument-specific effects in order to correct measured spectra of an analyte and to obtain the pure analyte-specific contributions. This also applies to measuring methods that compare the emission of luminophores with spectrally unlike emission spectra, such as for example the determination of fluorescence quantum yields, and for emission measurements at various excitation wavelengths. These instrument-specific effects are identified by the determination of what are called emission and excitation correction functions, which include the wavelength dependence and polarization dependence of the spectral sensitivity of the detection channels used (emission correction) and the wavelength dependence and polarization dependence of spectral illuminance at the sampling location and the intensity of excitation light (excitation correction).
Simple and restorable calibration of luminescence-measuring systems can be effected with certified physical transfer standards, that is, with a certified radiation-intensity-per-unit-area standard, a standard lamp (typically for the emission channel) and/or a certified receiver standard (typically for the excitation channel). Alternatively, chemical transfer- and so-called fluorescence standards with (ideally certified) spectrally corrected, that is, instrument-independent emission and/or excitation spectra can be used. The restorable and ideally certified (physical or chemical) transfer standards should insofar as possible be measurable under conditions identical to those of typical samples (for example in like sample containers/formats, with like polarizer settings, filters and reducers, as well as like settings for monochromator slot width, photomultiplier voltage, sampling interval and integration time or scanning speed) and be suitable for as many as possible different types of instruments, formats and measurement geometries, and cover a broad spectral region, typically UV/vis, vis/NIR and/or UV/vis/NIR. With increasing use of the NIR spectral region for a great variety of fluorescence applications (for example, optical imaging; 1st diagnostic window of about 650 to 900 nm and 2nd diagnostic window of about 1300 to 1500 nm), standards for the NIR spectral region are becoming increasingly important.
A prerequisite for suitability as a chemical transfer standard and reference material is problem-specific dye selection. This implies for example broad, smooth and unstructured emission spectra in the case of spectral emission standards and broad, smooth and unstructured absorption and excitation spectra in the case of excitation standards. For chemical transfer standards, their corrected emission and/or excitation spectra and preferably measurement uncertainty must in addition be known.
The restorable calibration of luminescence-measuring systems in the UV/vis/NIR spectral region requires the combination of a plurality of chromophores with (ideally certified) corrected fluorescence spectra which are adapted to each other. This includes, in particular, in addition to broad, rising as flatly as possible, unstructured bands of points of intersection of adjacent spectra at sufficiently high fluorescence intensities, so as to ensure linking of individual correction curves to a total correction function with as little as possible measurement uncertainty. There are only a few examples of the application of chromophore combinations to the determination of emission correction in the UV/vis/NIR spectral region. Chromophore-containing polymer films (NIST, certified emission spectra), a combination of the emission standard quinine sulfate dihydrate (SRM 936a) with cell-like chromophore-doped glasses and emission standard solutions are examples of emission standard combinations (for example, J. A. Gardecki, M. Maroncelli, Appl. Spectrosc. 1998, 52, 1179). In addition, chromophore-containing polymethyl methacrylate (PMMA) blocks in cell form are available as emission standards (Starna, Optiglass) and standard solutions (Invitrogen, formerly Molecular Probes). A kit of spectral fluorescence standards, which permits emission and absorption correction in a broad spectral region, is disclosed in
DE 10 2004 044,717 A. According to the later application DE 10 2008 040,513.2, the covered spectral region is capable of being broadened in the NIR region by combination with a long wave-emitting cyanine compound as the spectral fluorescence standard.
In addition, statistical methods for linking the correction functions obtained for the individual emission standards to a total correction function are [disclosed] in the Gardecki source and in DE 10 2004 044,717 A, mentioned above.
All known standard combinations always present a combination of unique standards, each standard often having to be excited at a different excitation wavelength for emission. This means that each component of a standard set must be measured separately, and each measurement supplies an emission correction curve for a limited spectral region of at most 150 to 200 nm (Δλ in the vis region) in the case of chromophore-based standards (for example quinine sulfate), which corresponds to the emission region of the respective standard and chromophore. These standard-specific correction curves must be combined in a subsequent step by evaluated mathematical procedures into a total correction curve, so as to cover a broad spectral region. Adaptation of the emission intensities of various standards to each other, which is necessary in for example the case of greatly varying extinction coefficients and fluorescence quantum yields as well as on the instrument side in the case of greatly varying spectral sensitivities, can only be effected by the adaptation of chromophore concentrations for liquid standards. In the case of solid standards, adaptation typically is no longer possible.
Standards for the Testing of Instrument Performance and Long-Term Stability of Instruments (Day-To-Day Intensity Standards)
Standards for the testing of instrument performance and long-term stability of instruments (so-called day-to-day intensity standards) are necessary in order for example to identify instrument drift caused by the aging of optical and optico-electronic structural parts and to permit comparability of measurements of relative fluorescence intensities carried out on different days. The availability of such standards is, in addition, very important for the regular performance of instrument tests and examinations necessary in connection with laboratory accreditations per ISO 17025. The most frequent and at present single established test for testing instrument performance and long-term instrument stability is the so-called Raman test. For this, non-fluorescent ultrapure water is irradiated at 350 nm, and the intensity of Raman scattering at 397 nm is measured. This test is only suitable for the UV spectral region.
In addition, it is known that variation of the intensity of emitters may be realized by their combination with an absorber. For this, quinine sulfate emitting broadband in the vis spectral region, for example, has been combined with a broadband-absorbing absorber (S. A. Tucker et al., J. Chem. Ed. 1992, 69(1), A8-A12) or a comparably narrow band-emitting fluorophore with a narrow-band absorber (R. Giebeler et al., J. Fluoresc. 2005, 15(3), 363-375).
Intensity Standards
Fundamentally, every luminescence technique supplies only relative intensities, if all photons emitted after chromophore excitation are not detected, as is the case, for example. in Ulbricht ball-measuring systems. For a quantification of intensity measurements, in most cases, either a correlation with the concentration of the emitter to be quantified is necessary (for example, by plotting a calibration straight line) or the use of an emitting reference system, i.e., an intensity standard. This may be by luminescence methods, for example fluorescence quantum yield standards (typically dye solutions of known fluorescence quanta yield) as well as calibration slides (microarray technology) or dye-labeled fluorescing particles, as in the case of flow cytometry, in which the emission intensity of the particles is previously quantified by comparing fluorescence measurements with the fluorophores used in the form of MESF units (measurable equivalents of soluble fluorophores). Another approach, derived from fluorescence microscopy, uses one or more fluorescing reference surfaces, where in as identical as possible a microenvironment a like fluorophore is used for the standard that is also to be quantified, so as to guarantee as similar as possible emission properties of sample and standard. The principle of signal referencing (signal ratioing) is common in fluorescence sensory analysis. Signal referencing can be realized in that the measured target-sensitive relative fluorescence intensity of a target-sensitive monochromatically emitting sensor molecule is referred to a second dye, which emits spectrally distinctly separately from the first fluorophore and the emission intensity of which is independent of the target. Alternatively, dual emitting sensor molecules, which, in the presence of the target exhibit very strong spectral shifts of their absorption (ratioing in excitation) or emission, or FRET systems, are used for this. Systems that can be adapted by the combination of various optical components with reference to their spectral properties to the respective problem, and the intensity of which can be adjusted or even varied problem-adapted by as simple as possible means, are desirable here. The combination of a fluorescing and an absorbing component, which serves for reduction or for the structuring of emission, or a reflecting component, is known here. For example, the combination of LEDs with reducers or the combination of emitters and absorbers for the calibration of microtiter plate readout instruments (R. Giebeler et al., J. Fluoresc. 2005, 15(3), 363-375) or an LED slide for fluorescence microscopy (I. T. Young, Proc. SPIE 1983, 38, 326-335) is well known. However, only the spectral properties or the intensity of a single fluorophore are varied there.
Intensity standards, the spectral emission properties and/or emission intensity of which can be adjusted and controlled as simply as possible without varying the chemical composition or the concentration, would be desirable, for example for application as a relative reference system for the ratiometric measurement of fluorescence intensities, for the detection of instrument drift and for the quantification of intensity-based fluorescence measurements. At the present time, a standard must be specially developed and evaluated for each application or for each desired spectral behavior or emission profile.
Determination of the Linearity Region of Luminescence Detection Systems
Knowledge of the linearity region of the detection systems used is a prerequisite for the determination of emission correction functions of luminescence-measuring systems and for any quantitative luminescence analytics. In the case of common detectors like PMTs and CCD systems, this depends upon the detection wavelength. At present, there is no uniform procedure for this. Dyes that are suitable for determination of the linearity region of fluorescence detection systems are characterized in particular by as little as possible overlapping of absorption and fluorescence. This requires that, at extinctions of up to about 0.1, emission spectra (1-cm cell) still be concentration-independent. Also generally advantageous are smooth unstructured emission spectra that cover as great as possible an emission spectral region.
One method, which is based upon an emission standard (quinine sulfate dihydrate), is described in ASTM E 578-83. The excitation and emission wavelength regions for which the method can be used, however, are limited by the absorption and emission spectrum of quinine sulfate dihydrate, so that the linearity region for the entire spectral region of interest cannot be determined. In addition, integral measuring fluorescence instruments, such as, for example, filter fluorometers, many microtiter plate readout instruments and scanners for optical imaging, are limited in the selection of excitation and emission wavelengths by the excitation light sources used (laser or lamp with excitation filter) and integral detection (emission filter and detector). Further, NIST-certified fluorescin solutions (SRM 1932) and the Molecular Probes company's fluorescin solutions (Fluorescin NIST-Traceable Standard F 36915) are available. Because of the comparatively narrow absorption and emission spectrum of fluorescin, these are only usable in a narrow vis spectral region. Also known are “Rediplate Microplate Intensity Standards” (Invitrogen), consisting of a plurality of fluorophores in solutions, which are problematic because of the great overlapping of the absorption and emission spectra, and “Fluorescence Reference Standards” (MATECH), which comprise chromophore-doped solid matrices, integrated in microtiter plates, for a variety of excitation and emission wavelengths in the UV/vis spectral region and thus are only suitable for microtiter plate readout instruments. Variation of concentration and there-fore of fluorescence intensity for problem-specific adaptation by the user is not possible.
At the present time, the concentration of the fluorophore must be varied for determination of the linearity region of luminescence detection systems. In addition, only a comparatively narrow spectral region is covered per measurement and per chromophore.
A standard that permits simple and rapid determination of the linearity of the detection system [in] a broad spectral region, preferably in the entire spectral region of the instrument (multifunctional reducer), with only a few measurements and ideally with a single standard, would be desirable.
Testing of Wavelength Accuracy and Spectral Resolution
For high-resolution spectrofluorometers, atomic emission lines of gas discharge lamps, which contain mixtures of gases such as neon and mercury, are typically used, so as to cover as great as possible a spectral region. For luminescence-measuring instruments such as microtiter plate readout instruments or spectral resolving micro-scopes or spectral resolving imaging systems for optical imaging, which have a smaller resolution of typically greater than 2 nm (so-called robust measuring instruments), wavelength accuracy and spectral resolution can also be obtained with narrow-band emitting chromophores (mixtures of a variety of lanthanide ions for example, in a glass matrix) or by the combination of one or more narrow-band absorbers with one or more emitters. There are examples of the combination of a fluorophore with an absorber, which have already been described above.
Scattering and Fluorescing Standards
Generally speaking, the measurement of fluorescence in scattering systems plays a great role in biomedical applications of fluorometry. This applies in particular to fluorometric investigations on tissues by methods of optical imaging, for example for the early diagnosis of disease-specific changes on the molecular level in the spectral region of from about 650 to 900 nm and in the second diagnostic window. So-called phantoms, which consist of an NIR fluorophore localized at one or more defined sites and a solid or liquid absorbing and scattering medium, are used as standards for this. Typically, Intralipid, Liposyn or microparticles (for example, silica, PMMA, polystyrene) inserted into a liquid or solid polymer matrix are used as a scatterer. The fluorophores used for the NIR region typically are symmetrical or asymmetrical cyanine dyes, such as indocyanine Green (ICG) or IR-125, diethylthiatricarbocyanine iodide (DTTCI), and IR-140, which are characterized by structured absorption and emission bands and a great overlapping region of absorption and fluorescence. Such systems are highly susceptible to internal filter effects (reabsorption).
Scattering fluorescence standards that permit a simple combination of any emitters with scatterers having variable scattering properties and optionally also with absorbers, so as to permit simple adaptation of the spectral emission and absorption properties of the standard to the respective problem, would be very desirable.